Protection of moist stratified squamous epithelia against damage from noxious luminal agents

ABSTRACT

The present invention is related to the protection of moist stratified squamous epithelia against damage from exposure to noxious luminal agents. Protection of moist stratified squamous epithelia against these noxious luminal agents is afforded by chemical compounds having one of the following reactive groups in their molecule: X--SO 3   - , where X represents oxygen or carbon, and XO 4   =  or X 2  O 7   = , where X represents an element from group VIb or sulfur of group VIa of the periodic table. Compounds that provide protection against injury to moist stratified squamous epithelia that illustrate the protective characteristic of these reactive species are the sulfonates, the sulfate esters and the tetrahedral-shaped divalent oxy-anions of the transition metals in group VIb or of sulfur. The reason for protection by these compounds is that they stabilize the intercellular junctions of moist stratified squamous epithelia so as to prevent the increase in permeability across the junctions that normally accompanies exposure to noxious luminal agents like HCl or N-acetylcysteine.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention, in general, relates to a method for protecting moiststratified squamous epithelia against damage from exposure to noxiousluminal substances.

In particular, this invention relates to:

I) the identification of a mechanism for protecting moist stratifiedsquamous epithelia against damage from exposure to luminal substancessuch as hydrochloric acid (HCl) and N-acetylcysteine. This is based onthe recognition that some of the noxious effects of luminal substanceson moist stratified squamous epithelia are a consequence of theirability to increase the permeability across the intercellular junctions(paracellular pathway), and that moist stratified squamous epithelia canbe protected from the noxious effect of luminal exposure to thesesubstances by stabilizing their intercellular junctions so as to blockthe increase in permeability through this route; and

II) the identification of 3 chemical reactive groups and three classesof chemical compounds derived from them that provide protection to moiststratified squamous epithelia against damage from luminal HCl orN-acetylcysteine by stabilizing their intercellular junctions so as toblock the increase in paracellular permeability that normallyaccompanies such exposure.

More specifically this invention relates to:

A) the identification of a new mechanism for protection of esophagealepithelia against acid injury and that is by stabilizing theirintercellular junctions so as to block the increase in paracellularpermeability that occurs with exposure to luminal acid; the ability toprotect by preventing the increase in paracellular permeabilityrepresents a new mechanism of "cytoprotection", cytoprotection bydefinition meaning a method of protecting against acid injury to tissuewithout inhibiting gastric acid secretion or altering luminal acidity,

B) the identification that the mechanism described in (A) also appliesto protection of esophageal epithelia against damage from luminalN-acetylcysteine and other noxious luminal substances that primarilydamage by increasing permeability through the paracellular pathway,

C) the identification that the mechanism described in (A) also appliesto protection of buccal and other moist stratified squamous epitheliaagainst damage from luminal HCl,

D) the identification of three chemical reactive groups and threeclasses of chemical compounds derived from them that with topicalapplication protect esophageal epithelia against damage from luminal HCIby stabilizing their intercellular junctions so as to block theHCl-induced increase in paracellular permeability.

The three reactive groups are:

a) X-SO₃ ⁻, where X represents an oxygen or carbon linkage covalently orionically bound to an organic or inorganic molecule, and thetetrahedral-shaped divalent oxy-anionic groups:

b) XO₄ ⁼ and

c) X₂ O₇ ⁼, where X represents an element from group VIb of the periodictable or sulfur from group VIa covalently or ionically bound to anorganic or inorganic molecule.

The three classes of chemical compounds derived from them are:

a) the sulfonates, e.g. 4-acetamido-4'-isothiocyano-2,2'-stilbenedisulfonate (SITS), 8-anilino-naphthalene-1-sulfonate (ANS),dinitrodisulfonic acid stilbene (DNDS), sulfonazo III,4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), bromphenol blue,

b) the sulfate esters, e.g. sucrose octasulfate, dextran sulfate, and

c) the tetrahedral-shaped divalent oxy-anions, e.g. sodium chromate,sodium dichromate, sodium molybdate, sodium tungstate, sodium sulfate,

E) the identification that the reactive groups and classes of compoundsreferred to in (D) also protect esophageal epithelia against damage fromluminal N-acetylcysteine and other luminal agents that damage byincreasing paracellular permeability,

F) the identification that the reactive groups and classes of compoundsreferred to in (D) also protect buccal epithelia against damage fromexposure to HCl and other luminal agents that damage by increasingparacellular permeability,

G) the identification that the reactive groups and classes of compoundsreferred to in (D), if shown to be non-toxic to humans, can be usedorally as a treatment to prevent reflux esophagitis or its symptoms(e.g., heartburn),

H) the establishment of a technique using the Ussing chamber and voltageclamp for identifying agents capable of protecting esophageal, buccaland other moist stratified squamous epithelia against luminal HCl,N-acetylcysteine and other noxious luminal agents that damage byincreasing the permeability through the paracellular pathway.

2. Background Information

Reflux esophagitis is a chronic disease that results from repeated andprolonged contact of esophageal epithelium with gastric acid (Richter J.E., Castell DO. Gastroesophageal Reflux: Pathogenesis, Diagnosis, andTherapy. Ann Intern Med 1982; 97-103). This represents one of the mostcommon illnesses in humans as attested to by the fact that there isalmost universal appreciation of its characteristic symptom,"heartburn". It has been reported that 7% of the U.S. populationexperience heartburn on a daily basis and that 36% experience it atleast once per month (Nebel TO, Fornes MF, Castell DO: Symptomaticgastroesophageal reflux: Incidence and precipitating factors. Am J DigDis 1976; 21:953). In general terms reflux esophagitis and its majorsymptom, heartburn, develop as a result of the chemical damaging effectsof gastric acid on the esophageal epithelium, and progression of thisdamaging action can lead to esophageal ulceration with bleeding,esophageal obstruction due to stricture formation and the development ofa Barrett's esophagus (i.e. replacement of the squamous epitheliallining by a metaplastic columnar epithelium), the latter a premalignantlesion. For these reasons reflux esophagitis is a serious disease.

There are two general approaches to treatment of reflux esophagitis. Thefirst and thus far most successful is to reduce gastric acidity. This isusually accomplished by inhibiting HCl secretion with either agents likecimetidine that block the parietal cell H₂ -receptor or with agents likeomeprazole that block the parietal cell enzyme H-K ATPase. Although theformer have been only moderately successful, the latter which haverecently been marketed reportedly heal up to 95% of patients with refluxdisease. However the relapse rates when therapy is stopped are high forboth types of agents, and that for omeprazole is 80% within six monthsof stopping therapy. Further although patients may respond toretreatment or be prevented from relapse by continuing the drug, thereis concern about the safety of omeprazole when used long term. This isbecause rats treated with omeprazole for long periods have developedgastric carcinoid tumors and these are believed to be secondary to therebound hypergastrinemia associated with potent acid suppression. Forthis reason there remains a need for therapeutic agents that can preventrelapse of reflux symptoms and esophagitis following treatment with themore potent acid-suppressing agents.

The second approach to treating reflux esophagitis involves using agentsthat enhance one of the intrinsic defenses of the esophagus. For examplebethanecol and metoclopromide have been used because of their abilitiesto increase the contractility of the lower esophageal sphincter (LES).This may theoretically be beneficial because the LES is the majorbarrier to reflux in humans. In practice however these agents have notbeen very effective.

Another defense mechanism that may be enhanced is that of the tissue'sintrinsic resistance to acid digestion, and agents that increase "tissueresistance" without inhibiting gastric acid secretion or luminalbuffering have been referred to as "cytoprotective". The inventors'laboratory has been interested in identifying compounds that protectesophagus against acid damage by a cytoprotective action on theepithelium. Such agents, the inventors found, can be identified in vitroby mounting esophageal epithelia in Ussing chambers hooked to voltageclamps and showing that an agent prevents the reduction in the tissue'selectrical resistance (R) upon exposure to luminal HCl. The ability ofan agent to block the HCl-induced decline in R confers protection onesophageal epithelia because the inventors had previously shown that theHCl-induced decline in R, reflecting an increase in tissue permeabilitythrough the intercellular junctions (paracellular pathway), precedes thedevelopment of cell necrosis (Orlando, R. C., D. W. Powell, and C. N.Carney. Pathophysiology of acute acid injury in rabbit esophagealepithelium. J. Clin. Invest, 68:286-293, 1981 and Orlando, R. C., J. C.Bryson, and D. W. Powell. Mechanisms of H injury in rabbit esophagealepithelium. Am J. Physiol, 246(Gastrointest. Liver Physiol. 9):G718-G724, 1984). Additional work by the inventors indicates that cellnecrosis occurs after the increase in paracellular permeability becauseit is the latter that allows hydrogen ions to enter the epithelium at asufficient rate to overcome intercellular buffering by serosalbicarbonate (Tobey, N. A., D. W. Powell, V. J. Schreiner and R. C.Orlando. Serosal bicarbonate protects against acid injury to rabbitesophagus. Gastroenterology 96:1466-77, 1989). The inability to bufferthe increased amounts of hydrogen ions eventually leads to cell death byacidifying the region adjacent to the basolateral cell membrane, thebasolateral cell membrane unlike the apical cell membrane being unableto tolerate even modest lowering of bathing solution pH without damageto the cell (Tobey NA, Orlando RC. Comparative sensitivity of rabbitesophageal epithelium to serosal versus luminal acid. Gastroenterology1989; 96:A512).

Sucralfate is a cytoprotective drug developed by Chugai in Japan andmarketed in the U.S. by Marion Laboratories. It has been used fortreatment of duodenal ulcer in the U.S. and more recently has beenstudied as a possible treatment for reflux esophagitis in humans (WeissW., Brunne H., Buttner G. R., et al. Treatment of reflux esophagitiswith sucralfate. Dtsch Med Wochenschr 1983; 108:1706). Although theefficacy of sucralfate in humans with reflux esophagitis has not beendramatic, the inventors experiments with sucralfate in esophagealepithelia mounted in Ussing chambers suggested that it contained apotent cytoprotective compound, and that this compound was sucroseoctasulfate (Orlando RC, N. A. Turjman, N. A. Tobey, V. J. Schreiner, D.W. Powell. Mucosal protection by sucralfate and its components inacid-exposed rabbit esophagus. Gastroenterology 1987; 93:352-61).Additional experiments also lead the inventors to recognize that theessential component responsible for the cytoprotective property of bothsucrose octasulfate and sucralfate was the presence of sulfate ionswithin the molecules (Tobey, N. A., R. C. Orlando, V. J. Schreiner, D.W. Powell. Cytoprotective effect of sulfate ions in acid-exposed rabbitesophagus. Am J Physiol 1986; 251:G 866-869). Also noteworthy was thatprotection by sucrose octasulfate and sulfate ions in vitro using theUssing chamber-voltage clamp technique were validated by showing thatthese same compounds exert protection in vitro in resected specimensfrom human esophagus (un published observations) and in vivo using as amodel the acid-perfused rabbit esophagus (Orlando RC et al.Gastroenterology 1987; 93:352-61 and Tobey NA et al. AM J Physiol 1986;251:G866-869).

Although the mechanism by which inorganic sulfate ions protected againstacid damage to esophageal epithelium was unknown and initiallyconsidered to be unique (Tobey NA et al. Am J Physiol 1986;251:G866-869), a chance occurrence lead us to identify that4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS) alsoprotected Ussing-chambered esophageal epithelium against luminal HClinjury in similar manner and at doses far lower than sulfate ions (TobeyNA, Schreiner V. J., Orlando R. C. Protection by SITS in acid-exposedrabbit esophagus. Gastroenterology 1988; 94:A461). Interestingly theinventors learned from the literature that sulfonates like SITS have astrong affinity for the same membrane receptor as sulfate ions and assuch inhibit sulfate transport in rat kidney cells (Fritzsch, G., G.Rumrich, K. J. Ullrich. Anion transport through the contraluminal cellmembrane of renal proximal tubule. The influence of hydrophobicity andmolecular charge distribution on the inhibitory activity of organicanions. Biochimica et Biophysica Acta 1989; 978:249-256 and K. J.Ullrich, G. Rumrich, S. Kloss. Contraluminal sulfate transport in theproximal tubule of the rat kidney. II Specificity: sulfate-ester,sulfonates and amino sulfonates. Pflugers Arch 1985; 404:293-299). Thissuggested to the inventors the possibility that sulfonates may protectesophageal epithelia against acid injury by binding to the same receptorin this tissue as sulfate ions. Similar reasoning was applied to thepossibility that tetrahedral-shaped divalent oxy-anions of elements fromgroup VIb of the periodic table (e.g. chromate) might protect againstacid injury to esophageal epithelia when it was learned from theliterature that these compounds also bind to sulfate receptors andinhibit sulfate transport in vesicles from placental epithelial cells(Boyd, CAR, D. B. Shennan. Sulphate transport into vesicles preparedfrom human placental brush border membranes: inhibition by trace elementoxides. J Physiol 1986; 379: 367-376). The present invention is based inlarge part on confirmation of this hypothesis--that is, that binding toa similar site in esophageal epithelium as sulfate ions confers on theseadditional agents the same protective properties against acid injuryobserved with sulfate treatment (see summary of invention below).Further the present invention also is broader based in that theprotective mechanisms and the identified protective agents also apply toprotection of esophageal epithelium against other luminal damagingagents (e.g. N-acetylcysteine) and to protection of other moiststratified squamous epithelia, e.g. buccal epithelium, against injuryfrom luminal HCl.

SUMMARY OF THE INVENTION

Accordingly the present invention relates to:

1) the identification of a general mechanism by which noxious luminalsubstances like HCl and N-acetylcysteine can be prevented from injuringmoist stratified squamous epithelia--that is, by stabilizing theintercellular junctions so as to prevent the increase in paracellularpathway permeability that accompanies exposure to these substances.

2) the identification of three chemical reactive groups and threeclasses of chemical compounds derived from them that when topicallyapplied protect moist stratified squamous epithelia against damage bystabilizing their intercellular junctions so as to block the increase inparacellular permeability that normally accompanies luminal exposure tonoxious substances such as HCl and N-acetylcysteine.

The three reactive groups are:

a) X-SO₃ ⁻, where X represents an oxygen or carbon linkage covalently orionically bound to an organic or inorganic molecule, and thetetrahedral-shaped divalent oxy-anionic groups:

b) XO₄ ⁼ and

c) X₂ O₇ ⁼, where X represents an element from group VIb of the periodictable or sulfur from group VIa covalently or ionically bound to anorganic or inorganic molecule.

The three classes of chemical compounds derived from them are:

a) the sulfonates, e.g. 4-acetamido-4'-isothiocyano-2,2'-stilbenedisulfonate (SITS), 8-anilino-naphthalene-1-sulfonate (ANS),dinitro-disulfonic acid stilbene (DNDS), sulfonazo III (sodium salt of3,6-bis-(o-sulfophenylazo)-4,5-dihydroxy 2.7-naphthalenedisulfonicacid), 4,4'-diisothiocyano-2,2'stilbene disulfonic (DIDS), bromphenolblue,

b) the sulfate esters, e.g. sucrose octasulfate, dextran sulfate, and

c) the tetrahedral-shaped divalent oxy-anions, e.g. sodium chromate,sodium dichromate, sodium molybdate, sodium tungstate, sodium sulfate,

Furthermore it is the object of the present invention to establish thatthere is more than one type of injury for which the above reactivegroups and protective compounds are capable of providing protectionagainst--e.g. HCl, N-acetylcysteine and others that injure by increasingparacellular permeability, and there is more than one type of moiststratified squamous epithelium that the reactive groups and protectiveagents are capable of providing protection to--e.g. esophagealepithelium, buccal epithelium and other moist stratified squamousepithelia, e.g. cornea, rumen, vagina, cervix, palate, gingiva, pharynxand tongue.

Moreover it is the object of the present invention to point out thatother compounds that have the identified reactive groups or fall intothe class of compounds identified above whether currently existing ornewly synthesized are likely to be protective, and that since protectionextends to HCl-induced injury to esophageal epithelium, may be effectiveagents for the prevention of reflux symptoms or esophagitis in humans.This latter possibility is particularly true because many of the agentshave the following clinically-attractive characteristics: activity atlow dose, rapid onset of action, topically active, long duration ofaction and potential for low toxicity because of poor absorption andlack of effect on baseline permeability and transport by the tissue.Notably these characteristics suggest that many of the agents are likelyto be at least as potent and potentially safer than those agentscurrently used for long term protection against acid damage toesophageal epithelium.

It is another object of the present invention to identify reactivegroups and classes of compounds that are more potent than sulfates forproviding protection against acid damage to moist stratified squamousepithelia. Furthermore, this invention provides both thestructure-activity relationships necessary for either the recognition ofother existing agents and/or the synthesis of new compounds with thecapacity to protect esophageal and buccal epithelia against acid injury,and the methodology (i.e. Ussing chamber-voltage clamp) to test theagents for protective activity in these tissues.

All publications mentioned are hereby incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention and many of the attendant advantages thereof arebetter appreciated by reference to the following detailed descriptionand the accompanying figures and tables.

FIG. 1 demonstrates that all of the readily available tetrahedral-shapeddivalent oxy-anions containing elements from periodic table VIb andsulfur from VIa provide protection against acid injury to rabbitesophageal epithelium mounted in Ussing chambers. This is illustrated bytheir ability, when added to the luminal bath, to prevent the decline intissue resistance (R) upon exposure to high concentrations of luminalHCl (pH 1.6). It is evident from the figure that the tetrahedral-shapeddivalent oxy-anions of the transition metals from group VIb have muchgreater protective effects than sulfate ions. Note: tissues were exposedto equimolar (38 mM) amounts of each agent before acidification of theluminal bath for 1 hour. In these experiments the agent remains in thebath during the acidification period. Also buffering does not accountfor protection by any agent because the luminal baths were titrated withHCl so that both experimental and control tissues had identical bathingsolution pH. The decline in R indicates increasing epithelialpermeability due to HCl damage, and thus prevention of the HCl-induceddecline in R reflects protection in this system. % Δ R=the 15 percentchange from preacidification values. Values are mean ± SE and the numberof experiments, n, is shown in parentheses for each agent.

FIG. 2 demonstrates the importance of the tetrahedral shape and divalentoxy-anion state to protection against acid injury to rabbit esophagealepithelium in the Ussing chamber. Using similar methodology as FIG. 1(i.e. 38 mM of the test agent), the results show that non-tetrahedralshaped divalent oxy-anions or a variety of monovalent anions in theluminal bath fail to protect against the HCl-induced decline inresistance (R). Note: the one significant change with NaSCN shows thatthis monovalent anion was actually more harmful to the tissue thancontrol.

FIG. 3 shows that the addition of 38 mM sodium molybdate, atetrahedral-shaped divalent oxy-anion, to the luminal bath has nosignificant effect on the baseline permeability of rabbit esophagealepithelia mounted in Ussing chambers as evident by the lack of change inbaseline R. It is evident that tissues exposed to molybdate areprotected against the decline in R upon luminal exposure to HCl (pH 1.6)for the duration of the exposure. Note: other electrical parametersmeasured but not shown (i.e. potential difference and short circuitcurrent) were also not significantly changed by the addition of the testagents to the luminal bathing solution. n=5

FIG. 4 shows that the lowest protective dose against acid damage torabbit esophagus in vitro for three of the tetrahedral-shaped divalentoxy-anions, i.e. sulfate, chromate and molybdate, is approximately 10-20mM. This experiment was performed similar to those of FIG. 1 inHCl-exposed rabbit esophageal epithelia mounted in Ussing chambers. R (%initial)=the percent change from preacidification values. Individualvalues are presented except for doses of 38 mM for which a mean of 5experiments is shown and for values at zero mM which are the controlswith no agent present.

FIGS. 5A and 5B show that tetrahedral-shaped divalent oxy-anions (38 mM)are capable of protection against acid damage to rabbit esophagealepithelium without being present in the luminal bath at the time ofacidification. This was done using a similar experimental design tothose of FIG. 1 except that tissues were only transiently exposed (15min or 1 min) to the agent before acidification of the luminal bath withHCl, (pH 1.6). Notably even after removing the agent, four of the sixtetrahedral-shaped divalent oxy-anions were able to protect against theHCl-induced decline in resistance (R).

FIG. 6 shows that protection against the HCl-induced decline inresistance (R) in vitro by a representative tetrahedral-shaped divalentoxy-anion, sodium molybdate (Na₂ MoO₄), predicted its ability to protectagainst acid damage to rabbit esophageal epithelium in vivo. In thiscase rabbit esophagi were cannulated and perfused for 1h with anisotonic solution of HCl (pH 1.0) after an initial 30 min perfusion witheither an isotonic molybdate-containing solution or an isotonic salinecontrol solution. Compared to controls, molybdate is shown to beprotective to esophageal tissues both morphologically by having asmaller area of injury identified on microscopy and functionally byhaving lower H+ efflux, lower permeability to mannitol and higherelectrical resistance (R). R and mucosal-to-serosal mannitol flux(J_(man) ms) were obtained from tissues mounted in Ussing chambers afterexposure to HCl in vivo. J^(H+) ms, H⁺ efflux, was measured for the invivo recirculated HCl solution. Values reported are mean ± SE, n=5.

FIG. 7 shows that 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate(SITS), a representative of the class of compounds known as sulfonates,when present in the luminal bath also protects against acid damage torabbit esophageal epithelium mounted in the Ussing chamber, and furtherthat this protection is afforded at doses that are 10-100 times lowerthan observed with compounds in the tetrahedral-shaped divalentoxy-anion group. Protection by luminal SITS is shown by its ability toblock the HCl-induced decline in electrical resistance (R) upon exposureof tissue to luminal HCl (pH 1.6) for 1 h. Control tissues , are exposedto Ringer solution. R (% initial)=percent change from preacidificationvalue. Values are mean ± SE, n=4-5.

FIG. 8 shows that the addition of 4 mM4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS), asulfonate, to the luminal bath has no significant effect on the baselinepermeability of rabbit esophageal epithelia mounted in Ussing chambersas evident by the lack of change in electrical resistance (R). Furtherdespite the lack of change in baseline R, it is evident that tissuesexposed to SITS are protected against the decline in R upon luminalexposure to HCl (pH 1.6) for the duration of the exposure. Note: otherelectrical parameters measured but not shown (i.e. potential differenceand short circuit current) were also not significantly changed by theaddition of the test agents to the luminal bathing solution. n=5

FIG. 9 shows that the protective effect of luminal4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS) at 4 mM isalso a property shared by a variety of sulfonated compounds with morethan one aromatic ring in the molecule. This is shown by the ability ofbromphenol blue, 8-anilino-naphthalene-1-sulfonate (ANS),dinitrodisulfonic acid stilbene (DNDS), sulfonazo III and4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), when added to theluminal bath, to protect against the decline in resistance (R) of rabbitesophageal epithelia exposed to HCl (pH 1.6) while mounted in Ussingchambers. Control tissues were exposed to normal Ringer only. R (%initial)=percent change from preacidification value. Values are means ±SE, n=3-7.

FIG. 10 shows that the disulfonates, ethane disulfonate and 1,3-benzenedisulfonate, which were not protective at 4 mM, were protective againstacid damage to rabbit esophageal epithelium at a higher dose of 40 mM.Note: even at this higher dose the aliphatic monosulfonate,mercaptoethane sulfonate, and the single ring aromatic monosulfonate,sulfanilic acid, were not protective. Protection is shown by the abilityto prevent the decline in resistance (R) of rabbit esophageal epitheliamounted in Ussing chambers and exposed to luminal HCl, pH 1.6, for 1 h.Control tissues were exposed to normal Ringer only. R (%initial)=percent change from preacidification value. Values are means±SE, n=3-4.

FIG. 11 shows that like some of the tetrahedral-shaped divalentoxy-anions, brief (1 min) transient luminal exposure to somerepresentative sulfonates (4 mM) can also protect against acid injury torabbit esophageal epithelium as shown by their ability to prevent thedecline in resistance (R) upon subsequent exposure to luminal HCl (pH1.6) for 1h.

FIG. 12 shows that 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate(SITS), a sulfonate, not only protects against acid damage to rabbitesophageal epithelium in vitro but also protects in vivo. In these invivo experiments rabbit esophagi were cannulated and perfused with 120mM HCl-20 mM NaCl in the presence or absence of SITS, 8 mM, for 1 h.Resistance and mucosal-to-serosal mannitol flux (J^(man) ms) wereperformed on tissues mounted in Ussing chambers after exposure to HCl invivo. Injury was scored as follows: 0=normal epithelium,1=intracellular/extracellular edema, 2=patchy intraepithelial cellnecrosis, 3=diffuse necrosis, and 4=ulceration (transmucosal necrosis).SITS is shown to protect the esophagus against acid damage in vivo bothmorphologically by a lower injury score on light microscopy andfunctionally by a lower permeability to mannitol and higher electricalresistance (R). Values are reported as mean ± SE, n=7.

FIG. 13 illustrates that transient (1 min) exposure of tissues to aprotective agent from either the sulfonate class,8-anilino-naphthalene-1-sulfonate (ANS), 4 mM, or the tetrahedral-shapeddivalent anion class, sodium dichromate, 38 mM, can protect even whenthe exposure to acid (HCl, pH 1.6 1 h) is delayed for up to 5 hours.Note: 1 min is used as the time for transient contact because it is asfast as the experiment can technically be carried out. Protection bythese compounds is likely to occur with much less time of contact.

FIG. 14 shows that 1 h luminal pretreatment with dextran sulfate (4 mM)but not an equimolar amount of dextran is protective against theHCl-induced decline in electrical resistance (R) of rabbit esophagealepithelia mounted in Ussing chambers. Note: this is another example ofprotection by compounds containing a sulfate ester linkage (O--SO₃ ⁻).Control tissues were exposed to normal Ringer solution before luminalacidification with HCl (pH 1.6) for 1 h. R (% initial)=percent changefrom preacidification value. Values are means ± SE.

FIG. 15 shows that 38 mM sodium molybdate, a tetrahedral-shaped divalentoxy-anion, or 4 mM 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate(SITS), a sulfonate, can not only protect against damage from exposureof rabbit esophageal epithelium to luminal HCl but protects againstdamage from exposure to luminal N-acetylcysteine, an agent that breaksdisulfide bonds. Protection against N-acetylcysteine is shown by theability of luminal pretreatment with either molybdate or SITS to preventthe decline in electrical resistance (R) of rabbit esophageal epitheliummounted in Ussing chambers and exposed to N-acetylcysteine (6%) for 1 h.

Table 1. Protection of dog buccal epithelium against luminal acid injuryby molybdate and SITS. The table shows that luminal 38 mM sodiummolybdate, a tetrahedral-shaped divalent oxy-anion, or luminal 4 mM4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS), asulfonate, can protect the moist stratified squamous epithelium of dogbuccal mucosa against acid damage. Protection is shown by the ability toprevent the decline in resistance (R) of dog buccal epithelium exposedto luminal HCl for 1 h in the Ussing chamber. Note: this low luminal pHis less well tolerated by buccal than by esophageal epithelium andconsequently protection while exhibited in buccal epithelium by theagents is much less dramatic.

                  TABLE 1                                                         ______________________________________                                        Effect of 15 min. pretreatment with protective agents on the                  electrical resistance of dog buccal mucosa mounted in the Ussing              chamber and luminally acidified (pH 1.5) with HCl for 1 h.                    PRETREATMENT  RESISTANCE (ohm · cm.sup.2)                            AGENT         PRE ACID   POST ACID                                            ______________________________________                                        SITS (4 mM)   747 ± 118                                                                             224 ± 25* (32 ± 6%)*                           Na.sub.2 --MoO.sub.4 (38 mM)                                                                505 ± 65                                                                              239 ± 44* (47 ± 7%)*                           Ringer Control                                                                              647 ± 101                                                                              90 ± 13  (15 +3%)                                ______________________________________                                         *p < 0.05 compared to paired control                                          Values reported as mean + SE                                                  n = 4                                                                         Numbers in parentheses express the post acid value as a percent of the pr     acid agent value.                                                        

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes:

1) a mechanism for protection of moist stratified squamous epitheliaagainst injury from exposure to luminal HCl and other noxious luminalsubstances such as N-acetylcysteine. The mechanism is by stabilizationof the intercellular junctions so as to block the increase inparacellular permeability that accompanies exposure to these noxiousluminal substances,

2) the identification of three chemical reactive groups and threeclasses of compounds derived from them that when topically appliedprotect moist stratified squamous epithelia against damage from luminalHCl and N-acetylcysteine by stabilizing their intercellular junctions soas to block the increase in paracellular permeability that normallyaccompanies such exposure.

The three reactive groups are:

a) X-SO₃ ⁻, where X represents an oxygen or carbon linkage covalently orionically bound to an organic or inorganic molecule, and thetetrahedral-shaped divalent oxy-anionic groups:

b) XO₄ ⁼ and

c) X₂ O₇ ⁼, where X represents an element from group VIb of the periodictable or sulfur from group VIa covalently or ionically bound to anorganic or inorganic molecule.

The three classes of chemical compounds derived from them are:

a) the sulfonates, e.g. 4-acetamido-4'-isothiocyano-2,2'-stilbenedisulfonate (SITS), 8-anilino-naphthalene-1-sulfonate (ANS),dinitro-disulfonic acid stilbene (DNDS), sulfonazo III,4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), bromphenol blue,

b) the sulfate esters, e.g. sucrose octasulfate, dextran sulfate, and

c) the tetrahedral-shaped divalent oxy-anions, e.g. sodium chromate,sodium dichromate, sodium molybdate, sodium tungstate, sodium sulfate,and

3) the likelihood that the protection afforded by the classes ofcompounds illustrated in (2), other existing or newly synthesizedcompounds in these classes or with the designated reactive groups arelikely to have clinical applicability for the prevention of refluxsymptoms and esophagitis in humans because they:

a) act topically and so can be administered orally,

b) have a rapid onset of action (≦1 min),

c) have a long duration of action (≧5 h),

d) can be administered in low dose (0.1-4 mM for sulfonates/sulfateester and 20-40 mM for tetrahedral-shaped divalent oxy-anions, and

e) have the potential for low toxicity because of poor absorption and,despite avid tissue binding, lack of effect on baseline permeability ortransport of the tissue. Note: poor absorption was shown by fluxes ofradiolabelled ³⁵ SO₄ ⁼ and ³ H-DIDS; for sulfate, n=4, the values were0.0036±0.002 μmol/h·cm² and in two experiments with DIDS the values were0.0003 and 0.0005 μmol/h·cm².

The new discoveries found during the current investigations that formthe basis of the present invention are those described in (1), (2), and(3) above.

Furthermore the classes and the agents described above are more potentprotectors against acid injury to esophageal epithelia than thepreviously reported sulfate ions. This may be a result of more avidbinding to the same receptor in esophageal epithelium as sulfate sinceit is evident that many of these same compounds bind to sulfatereceptors inhibiting sulfate transport in other tissues, e.g. kidney andplacenta. Some additional experimental data in support of the protectiveagents being bound to protein within esophageal epithelium was shown byexposing the tissue briefly to luminal radiolabelled-sulfate (³⁵ SO4=)or radiolabelled-sulfonate (³ H-DIDS) and by the inventors being unableto subsequently extract any of the radiolabelled-agent from the tissueafter homogenization and extraction with methanolchloroform, a lipidsolubilizing procedure (Folch J., Lees M., Stanley G. H. A simple methodfor the isolation and purification of total lipids from animal tissue. JBiol Chem 1957; 226: 497-509).

Proof that the above agents protect against acid injury to esophagealepithelium was carried out in rabbit esophagus both in vitro and invivo. In vitro studies consisted of mounting healthy rabbit esophagealepithelia in Ussing chambers, exposing them on the luminal surfacecontinuously or transiently to a test agent and monitoring the change inelectrical resistance, a marker of tissue permeability, upon luminalacidification with HCI (see methods for details). In healthy controltissues HCl exposure reduced resistance with time while protection by anagent is shown by its ability to prevent the decline in resistance. Thefact that the decline in resistance indicates increasing permeability ofthe tissue and the reasons for it being a forerunner to the developmentof tissue necrosis has been discussed above.

Compounds of the sulfonate, sulfate ester and tetrahedral-shapeddivalent oxy-anion class were shown to be protective either when presentin the luminal bath at the time of acidification or when tissues wereonly briefly (1 min) exposed to the compounds before acidification ofthe luminal bath. This was evident in their ability to prevent theHCl-induced decline in resistance shown to occur in the simultaneouslystudied and electrically (by resistance) paired control tissues. Inaddition with some compounds even with brief tissue exposure (1 min)protection against acid injury could be shown and protection with briefexposure shown even when luminal acidification was delayed for 5 hours.This establishes that protective compounds in these two classes can havea long duration of action even with minimal tissue contact time.

Protection by these compounds was not only shown in vitro by the abilityto prevent the decline in resistance of HCl-exposed rabbit esophagealepithelia in the Ussing chamber but was confirmed in vivo using theHCl-perfused rabbit esophagus model (see methods for details). In theseexperiments tissues were exposed in vivo to an isosmotic solutioncontaining a protective agent or to an isosmotic control solution priorto being perfused with HCl for 1 h in vivo. After exposure a number ofparameters were used to define if tissues were damaged or protectedincluding: gross inspection, light microscopy, electrical resistance andpermeability to mannitol (mannitol flux), the latter two parametersobtained by mounting a section of HCl-perfused epithelium in the Ussingchamber. For two classes of compounds, the sulfonates (SITS the testagent) and tetrahedral-shaped divalent oxy-anions (sodium molybdate thetest agent) protection was documented by all criteria when compared tocontrol animals. This confirmed that protection in vitro, that is byblocking the HCl-induced decline in resistance, was indeed predictive ofa compounds ability to protect in vivo. Further and supportingprotection as a general characteristic of both tetrahedral-shapeddivalent oxy-anions and compounds like the sulfonates bearing the X--SO₃⁻, reactive group, this same sequence has been previously reported (seeprior references) for sodium sulfate, sulfate ions being atetrahedral-shaped divalent oxy-anion (XO₄ ⁼ group), and for sucroseoctasulfate which contains the sulfate ester O--SO₃ ⁻ group.

These classes of protective agents were also shown to protect againstacid injury to another type of moist stratified squamous epithelia fromanother species, that is the dog buccal epithelium. This indicates thatprotection against acid injury by tetrahedral-shaped divalent oxy-anionsand X--SO₃ ⁻ containing compounds of which sulfonates represent oneimportant group are a general phenomena extending to other moiststratified squamous epithelia (e.g cornea, cervix, vagina, tongue,gingiva, palate, pharynx, rumen of animals). A recent example to furthersupport this comes from a report by Yanagisawa and colleagues(Yanagisawa T., Wakabayashi S., Tomiyama T., Yasunami M., Takase K.Synthesis and anti-ulcer activities of sodium alkylazulene sulfonates.Chem Pharm Bull 1988; 36:641-647). These investigators were developingcompounds for use in the treatment of peptic ulcer, basing theirresearch on the known protective agents "guaiazulene" and itshydrophilic derivative, guaiazulene sodium sulfonate (Okabe S., TakeuciK., H. and K. Takagi. Pharmacometrics 1975; 9:31). Interestingly, andsupporting the present inventors' concepts, they found that thesulfonate derivates were the most potent for protection againstulceration of the rat forestomach (Shay model)--the forestomach being anarea lined by stratified squamous epithelia. Further and in contrast tothe present inventors' work they attributed the mechanism of action ofthese compounds to the ability of guaiazulene and its derivates toinhibit the activity of the enzyme pepsin rather than, as suggestedherein, by the ability of the sulfonate groups to stabilize theintercellular junctions of moist stratified squamous epithelium so as toprotect them against injury by luminal HCl.

The present invention relates to the protection of the above moiststratified squamous epithelia against acid injury by administering theprotective compound in such a way as to make contact with the luminalsurface of the tissue. This includes such general methods ofadministration as topical application and perfusion via a tube for allmoist stratified squamous epithelia, oral ingestion for oropharyngeal,rumen and esophageal epithelium, mouth rinse (for oral epithelium), eyerinse (for cornea) and douche (for cervix or vagina).

While the present invention is directed specifically towards protectionof moist stratified squamous epithelia against acid injury by topicallyadministering a compound containing one of the reactive groups orbelonging to one of the classes of agents based on them, the presentinvention is equally applicable to protection of moist stratifiedsquamous epithelia against injury from other noxious luminal agents thatdamage the tissue similar to that of acid--that is by increasingjunctional (paracellular) permeability. This concept is demonstrated bythe ability of the defined cytoprotective compounds to protect rabbitesophageal epithelia against the decline in resistance upon exposure toluminal N-acetylcysteine. N-acetylcysteine is known to break disulfidebonds and presumably increases permeability through the paracellularpathway because the initial reductions in electrical resistance are notassociated with cell edema or necrosis (cell edema or necrosis wouldlikely occur if such large increases in permeability occurred across thecell membrane rather than the junction). The observation that thedecline in resistance is blocked by a cytoprotective compound that alsoprotects against the HCl-induced decline in resistance also supportsthat damage by acid and N-acetylcysteine occur at the same site, that isat the intercellular junctions. If injury to moist stratified squamousepithelia from infectious agents (e.g., bacteria, viruses, yeast orfungi), chemicals (e.g., lye, bleach, tobacco products) and/or otherluminal environmental conditions (e.g., heat, hypertonicity) occurs byincreasing the permeability through the intercellular junctions, thenthe presently described cytoprotective agents by virtue of theirstabilizing effect on the intercellular junctions would exert aprotective action.

Treatment by these compounds would consist of topical application to amoist stratified squamous epithelia of either an existing or newlysynthesized compound containing at least one of the defined reactivegroups defined in (2) above, but usually as shown for the most effectivesulfonates has more than one reactive group, that is R(XSO₃ --)_(n)Y_(n) ⁺ (where n is a positive integer, Y represents an element in groupIA of the periodic table, and X is an oxygen or carbon linkage betweenthe --SO₃ -- group and R, R representing an organic or inorganic groupcovalently or ionically bonded to X. The cytoprotective compounds wouldalso include any precursor compounds that when subjected to an acidic orother chemical environment are reduced to yield one of the definedreactive groups in (2).

Examples of cytoprotective compounds containing an --X--SO₃ --, reactivegroup, where X is a carbon as in the sulfonates or oxygen as in thesulfate esters include but are not limited to:4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonate (SITS),8-anilino-naphthalene-1-sulfonate (ANS), dinitro-disulfonic acidstilbene (DNDS), sulfonazo III, 4,4'-disothiocyano-2'2-stilbenedisulfonate (DIDS), bromphenol blue, sucrose octasulfate and dextransulfate.

Examples of cytoprotective compounds containing a tetrahedral-shapeddivalent oxy-anion of the transition metals in group VIb of the periodictable or sulfur in group VIa include but are not limited to: sodiumdichromate, sodium molybdate, sodium tungstate, sodium sulfate.

Moreover agents that can be used for prevention of acid injury to theesophageal epithelia in humans whether existing or newly synthesizedusing the configurations outlined above may be effective for preventionof reflux disease in humans.

In a preferred embodiment, a cytoprotective compound having one of thedefined configurations described above and shown to be safe for use inhumans would be administered orally to a subject with refluxesophagitis. An adult of average size would be expected to ingest inliquid or powder form the equivalent of 0.1-4 mM of the sulfonate orsulfate-ester type agents or 10-20 mM of the tetrahedral-shaped agentfrom two-to-four times per day (total does in milligrams/grams woulddepend upon the molecular weight of the compound). Contact of theesophageal epithelium even briefly with the ingested agent would then beexpected to prevent further acid damage to the tissue for many hours.

Methods

I. For tetrahedral-shaped divalent oxy-anions.

In vitro. White New Zealand rabbits weighing 8-9 lbs. were killed withan overdoses of pentobarbital sodium (60 mg/ml), and the esophagus wasremoved. The esophageal epithelium was stripped of its muscle layers andmounted in Ussing chambers. The luminal and serosal sides were bathedwith Ringer solution (in mM) 140 Na⁺, 119.8Cl--, 5K⁺, 25 HCO₃ --, 1.2Mg²⁺, 1.2 ca⁺⁺, 2.4 HPO₄ ⁻ , 0.4 H₂ PO₄ ⁻ (280 mosmoml/kg H₂ O, pH 7.4when gassed with 95% O₂ /5%, CO, maintained at 37° C.). Luminal andserosal solutions of similar volume and composition were in contact withcalomel and Ag-AgCl electrodes via agar bridges. The electrodes wereconnected to an automatic voltage clamp for measurements of potentialdifferences (PD) and short-circuit current (Isc). Tissues werecontinuously short-circuited except for brief intervals (2-3 sec) whenthe open circuit PD was read. Resistance (R) was calculated using Ohm'slaw from the open circuit PD and the Isc. After 45 min forequilibration, epithelia were paired by R (R within 25%). One of thepair was then exposed luminally to a sodium salt (38 mM) of atetrahedral-shaped divalent oxy-anion, a non-tetrahedral shaped divalentanion, or a monovalent anion. The other tissue was exposed toiso-osmotic sodium chloride (NaCl) as control. After 1 h of treatment,luminal solutions were titrated to pH 1.6 and an equimolar amount ofcholine chloride added to the serosal bath to balance osmolality and tolimit ionic diffusion to H⁺ alone. Controls required 60 mM HCl totitrate pH to 1.6. All other agents except the following requiredsimilar amounts of HCl to titrate the bath to pH 1.6: chromate 108 mM,tungstate 119 mM, molybdate 122 mM, sulfate 72 mM, and monohydrogenphosphate 118 mM. After initial titration, luminal pH for all agentsremained constant throughout the experiment. After luminalacidification, R was monitored for 1 h.

In vivo. White New Zealand rabbits weighing between 8-9 lbs. wereanesthetized with a 1:1 mixture of diazepam (5 mg/ml) and pentobarbitalsodium (60 mg/ml). The esophagus was cannulated in the neck and in theabdomen just above the esophagogastric junction. The two cannulas wereconnected via a Buchler polystaltic pump so that 50 ml of an HClsolution could be perfused and recirculated through the esophagus (37°C., 10 ml/min). Esophagi were initially perfused for 30 min with 38 mMNa₂ MoO₄ -90 mM NaCl (242 mosm/kg H₂ O) or an iso-osmotic solution ofsaline (150 mM NaCl) (284 mosm/kg H₂ O) as control. After theseperfusions, esophagi of all animals were flushed with 50 cc normalsaline. Esophagi were then perfused, using a recirculating system, for 1h with 120 mM HCl-20 mM NaCl, pH 1.0. After acid exposure, the perfusatewas collected for determination of volume and calculation of H⁺ content.H⁺ content was determined by titration of duplicate 10 ml aliquots to pH7 with 1 N NaOH using a Radiometer PHM82 pH meter (Radiometer,Copenhagen, Denmark). H⁺ efflux (J^(H+) m-s) (loss from the lumen) wasdetermined by measuring the difference between the amount of H⁺ insolution before and after the 1 h esophageal perfusion. H⁺ efflux wasreported in ueq h⁻¹ cm⁻² by dividing the total H⁺ lost from theperfusate by the area of esophagus exposed to solution. The area ofexposure was determined, after killing the animal with an overdose ofpentobarbital and excising the esophagus, by measuring the length andwidth of esophagus between the two cannulas.

After removal, the esophagus was inspected for gross lesions and asection from the distal third obtained for mounting in the Ussingchamber. This section was stripped of its muscle layers and mounted in10 mM mannitol-Ringer solution in the Ussing chamber for assessment ofpermeability by measurements of R and mucosal-to-serosal [¹⁴ C]mannitolflux (J^(man) m-s). [¹⁴ C]mannitol fluxes were performed by adding 10μCi of [¹⁴ C]mannitol to the luminal bath. After a 15 min equilibrationperiod, R was recorded, and five 15 min fluxes of [¹⁴ C]mannitol weredetermined. Flux data reported represent the mean for the five fluxperiods. The remainder of the HCl-exposed esophagus was fixed in 2%paraformaldehyde, 4% glutaraldehyde in 0.1N phosphate buffer, pH 7.4,cut into equal-sized sections and stained with hematoxylin and eosin forevaluation by light microscopy. Histology slides were coded and read bythe examiner with no knowledge of the treatment group. For each sectionthe presence or absence of necrosis was noted and when present, apercentage appended to reflect the linear extent of the epitheliumhaving necrosis.

Statistical significance was determined using Students's t-test and alldata were reported as the mean ± standard error (SE).

II. For the sulfonates and sulfate esters.

In vitro. White New Zealand rabbits weighing 8-9 lbs were killed with anoverdose of pentobarbital sodium (60 mg/ml), and the esophagus wasremoved. The esophageal epithelium was stripped of its muscle layers andmounted in Ussing chambers. The luminal and serosal sides were bathedwith Ringer solution (in mM) 140 Na⁺, 119.8 Cl⁻, 5.2 K⁺, 25 HCO₃ --, 1.2Mg²⁺, 1.2 Ca²⁺, 2.4 HPO₄ ²⁻, 0.4 H₂ PO₄ -- (280 mosm/kg H₂ O), pH 7.4when gassed with 95% O₂ -- 5% CO₂ maintained at 37° C. Luminal andserosal solutions of similar volume and composition were in contact withcalomel and Ag-AgCl electrodes via agar bridges. The electrodes wereconnected to an automatic voltage clamp for measurements of potentialdifference (PD) and short-circuit current (Isc). Tissues werecontinuously short-circuited except for brief intervals (2-3 s) when theopen circuit PD was read. Resistance (R) was calculated using Ohm's lawfrom the open circuit PD and the Isc.

To assess a compounds ability to protect rabbit esophageal epitheliaagainst acid injury, after equilibration (approximately 45 min aftermounting), tissues paired by R (R within 25%) were exposed luminally toa test agent (e.g. SITS) while the other served as untreated control.After 1 h, the luminal solutions were acidified with HCl to pH 1.6(note: some agents acted as weak buffers requiring from 0-40 mM more HClto reach pH 1.6 than others) and equimolar amounts of choline chlorideadded serosally to balance osmolality and limit ionic diffusion to H⁺alone. PD, Isc and R were monitored every 15 min through the experiment.

In some experiments, tissues were pretreated luminally with SITS for 1 hand the SITS-containing luminal solution replaced with normal Ringerprior to luminal acidification with HCl. Electrical parameters weremonitored every 15 minutes as described above.

Junction potentials were determined for all solutions used in theseexperiments. Solutions reflecting those present under experimentalconditions were placed in separate beakers and the beakers used in pairsto reflect luminal or serosal bathing solutions for tissues mounted inthe Ussing chamber. The results, however, showed that all junctionpotentials were insignificant being <1 mV and so were not corrected forin the presentation of results.

In vivo. Protection against acid injury was assessed using a perfusiontechnique. White New Zealand rabbits weighing 8-9 lb were anesthetizedwith a 1:1 mixture of diazepam (95 mg/ml) and pentobarbital sodium (60mg/ml) and strapped to an animal board covered with a heating pad tomaintain body temperature at 37° C. The esophagus was cannulated in theneck and in the abdomen just above the esophagogastric junction. The twocannulas were connected via a Buchler polystaltic pump so that 50 ml ofan HCl solution could be perfused and recirculated through the esophagus(37° C., 10 ml/min). Esophagi were perfused with saline for 30 minfollowed by 120 mM HCl-20 mM NaCl for 1 h (control) or saline+8 mM SITSfollowed by 120 mM HCl-20 mM NaCl+8 mM SITS. SITS - containingperfusates had approximately the same osmolality (276 vs 263 mosmol/kgH₂ O for controls) and Na content (36 mM vs 20 mM Na for controls) asthat of the saline-controls. After perfusion of the esophagus, therabbits were killed with an overdose of pentobarbital sodium. Theesophagus was removed and inspected for gross lesions. One section ofesophagus was stripped of its muscle layers and mounted in 10 mMmannitol-Ringer solution in the Ussing chamber for measurements of R andfor assessment of permeability by measuring the mucosal-to-serosal [¹⁴C]mannitol flux (J^(man) ms). [¹⁴ C]mannitol was added to the luminalbath and after a 15-min equilibration period, R was recorded, and five15-min fluxes of [¹⁴ C]mannitol were determined. Mean data for the fiveflux periods is reported. A second section of esophagus was fixed (2%paraformaldehyde, 4% glutaraldehyde, 0.1N phosphate buffer, pH 7.0) andstained with hematoxylin and eosin for evaluation by light microscopy.Lesions were recorded and scored by an examiner without knowledge oftreatment groups using the following system: 0=normal epithelium,1=intracellular/extracellular edema, 2=patchy intraepithelial cellnecrosis, 3=diffuse necrosis, and 4=ulceration (transmucosal necrosis).

Statistical significance was determined using Students's t-test forparametric data and the Wilcoxon signed ranks test for non-parametric(morphology) data. All data were reported as the means ± standard error(SE).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention.

What is claimed is:
 1. A method of protection of moist stratified squamous epithelia against damage from a noxious luminal substance primarily causing damage by increasing permeability through the paracellular pathway which comprises stabilizing epithelial intercellular junctions in a patient having exhibited the symptoms of gastroesophageal reflux or reflux esophagitis by administering to said patient so as to come into contact with said epithelia an amount sufficient for protecting said epithelia of a sulfonate so as to block the increase in paracellular permeability that occurs with exposure to said noxious luminal substance.
 2. The method according to claim 1 wherein said sulfonate is 4-acetamido-4°-isothiocyano-2,2'-stilbene disulfonate, 8-anilinonaphthalene-1-sulfonate, dinitro-disulfonic acid stilbene, sulfonazo III, 4,4'-diisothiocyano-2,2'-stillbene disulfonate, or bromophenol blue.
 3. The method according to claim 1 wherein said moist stratified squamous epithelia is esophageal epithelia or buccal epithelia.
 4. The method according to claim 1 wherein said noxious luminal substance is acid or N-acetylcysteine.
 5. The method of claim 1 wherein said sulfonate is sulfonazo III. 